ANTIMICROBIAL RESISTANCE

Penicillin was a huge breakthorugh that saved millions of lives, but a few years after it was put on the market, penicillin resistant S. aureus was found. In the 70’s resistant strains of H. influenza, gonorrhea, and shigella emerged. Antimicrobial resistance is now a worldwide problem. There has been a dramatic increase in antimicrobial resistant community acquired and nosocomial pathogens. Major risk factors include antimicrobial misuse and noncompliance. Currently there is MDR-TB, penicillin resistant pneumococci, malaria, cholera, MDR-gonorrhea, VRE and MRSA in the community, 60% of vsceral leishmaniasis are resistant along with 60% of hospital acquired infections.

Consequently there has been an increased mortality because usually the people who get these diseases are the ones in hospitals. There is also an increase in morbidity because the illness takes longer to be resloved and there is a greater chance for resistant organisms to spread to other people. Resistance means increased costs of care due to newer and more expensive drugs. The future is bleak as there are few new drugs on the horizon.

MOLECULAR GENETICS OF ANTIBIOTIC RESISTANCE

DEFINITIONS:

1. Plasmids  y’all already know about plasmids, plasmids can encode antibiotic resistance and virulence factors.

2. Bacteriophages  viral nucleic acid can be injected into the host cell

3. Transposable Elements  DNA segments that can insert themsleves into DNA molecules and leave. They can’t replicate by themselves and so must be on a replicon like a chromsome, bacteriophage, or plasmid. Can be trabsferred along a chromosme. There are 2 kinds: insertion sequences (IS elements) which have minimal genetic information for transposition and Transposons (Tn) which also have other genes such as antibiotic-resistance marker.

4. 4 Modes of introducing DNA into bacteria  these are transformation, transduction, conjugation, and transposition and have already been covered in previous lectures.

Genetic Mechanisms of Antiobiotic Resistance

1. Inherent (natural) resistance  pseudomonas are always resistant to penicillin.

2. Point mutations in the chromosomes  can alter target site of an antimicrobial agent.

3. Rearrangements  by all the methods we learned about

4. Foreign DNA  carried by all the methods mentioned above, this is the main mechanism for antibiotic resistance, especially plasmid mediated resistance. It is easily transmissable, highly stable, confers resistance to lots of classes of antibiotics at the same time. And is also associated with other virulence factors. An example of this is the spread of β-lactamase from enterobacteriaceae to H. influenza and N. gonorrhea.

Antibiotics have a strong selective pressure on the development of resistance by destroying the susceptible bacteria but allowing the resistant ones to proliferate.

BIOCHEMICAL MECHANISMS OF ANTIBIOTIC RESISTANCE

1. Drug Inactivation 

   1. β-lactamases  inactivate β-lactams by splitting the amide bond if the ring.

   • There are over 340 different types that can be inducible (level of production varies with the induction potential of specific agents, strong inducers are imipenem, ampicillin, cefoxitin, and clavulanate; modate inducers include sulbactam, and weak inducers are ceftriaxone, ureidopenicillins, and tazobactam) or Constitutive (constant level of production).
     

   • In Gram +ve bacteria they are excreted into the environment; all hospital isolated staph and 50-80% of community aquired staph can make these enzymes.
     

   • In Gram –ve they are excreted into the periplasm, these are found mainly in the Enterobacter species.
     

   • They are classified by genetic or biochemical properties. TEM1 accounts for 75-80% of plasmid-mediated resistance worldwide and is found mainly in influenza, Slamonella, Shigella, and Gonorrhea.
     

   • ESBL give resistance to all penicillins and cephalosporines and is a problem in lots of hospitals.

2. Β-lactamases can be classified into 4 groups:

1. Group A has serine at the active site, constitutive, usually found on plasmids. Preferred hydrolysis of penicillin, the group includes TEM-1, SHV-1, penicillinase.

2. Group B are metalloenzymes with Zn at the active site, hydrolyze carbapenems, include IMP-1

3. Group C has serine at the active site and is found on the ampC gene. It is inducible and includes enzymes such as MIR-1.

4. Group D has serine at the active site, hydrolyze Oxacillin, OXA-1.

Another way of classifying β-lactams is the Bush-Jacoby-Medeiros functional classification scheme. Each group has 20-80 different enzymes.

2. Aminoglycoside modifying enzymes  there are more than 24 and are coded for on plasmids or the chromsome. There are 3 reactions – N-acetylation, O-nucleotidylation, and O-phosphorylation.

3. Chloramphenicol acetyltransferase

4. Erythromycin esterase

2. Reduced Target Affinity 

1. Alteration of target enzymes  altered PBP give a decreased affinity for β-lactams. Methicillin resistance S. aureus (MRSA) and MRSE (S. epidermidis) are formed by a PBP2a encoded by mecA gene. It is capable of cell wall synthesis in the absence of other PBPs has low affinity for all β-lactams. Altered DNA gyrase gives resistant to quinolones. Altered synthetase gives resistance to sulfonamides.

2. Alteration of ribosomal targets  MLS resistance is conferred by genes that encode 8 classes of enzymes that demethylate adenine residue on 23S rRNA of the 50S subunit. Conformational changes in the ribosome results in a decreased affinity and leads to coresistance to all MLS antibiotics. They are plasmid or chromosomal encoded. Aminoglycoside resistance is caused by mutations of S12 protein of the 30S subunit.

3. Alterations of cell wall precursor targets  Glycopeptides bind to D-ala-D-ala at the end of the peptidoglycan precursors and prevent further transglycosilation. Resistance happens when they turn it into D-ala-D-lac. The vancomycin don’t fit anymore.

4. Reduced permiability  Penicillin is effective against gram+, but not gram _ due to the outer membrane. LPS impedes the entry of hydrophobic antibiotics. Some bacteria can change their production of porins. The bigger the antibiotic molecule, the more negative charges, the greater the degree of hydrophobicity, the less likely it is to penetrate through the OM. Mutations in porins can lead to increased resistance to β-lactams, aminoglycosides, quinolones.

1. Antibiotic Efflux  There can be active efflux of antibiotics by the inner membrane. This is the major mechanism of resistance in Gram- bacteria to tetracycline. This is found in E.coli, shigella, and pseudomonas.

2. Bypass of Antibiotic  Folate auxotrophs can use their own folates so they’re not susceptible to sulfonamides and trimethoprim.

We can test for susceptible bacteria by the MIC (Minimal Inhibitory Concentration)  the lowest concentration of a specific antimicrobial agent that inhibits the test organism. The MBC (Minimal Bactericidal Concentration) is the lowest concentration of a specific antimicrobial agent that kills the test organism.

Disk diffusion susceptibility testing  a disk with antibiotics is put on an agar plate after the test organism has been put on it. The diameter of the zone of inhibition after incubation for 16-18 hours. The zone diameter interpretive breakpoints are based on the inverse correlation of zone diameter with MIC.

Agar dilution testing is cost effective if a large number of isolates are tested at the same time. Many different organisms can be tested simultaneously on each plate.

E test is a new procedure that uses a strip impregnated with antimicrobial agent and applies it to a plate. The plate is examined for growth.

Antibiotic combinations are used for life-threatening infections, prevention of emergence of bacterial resistance, polymicrobial infections, decreased toxicity, and enhanced antibacterial activity. Synergy is when the combined drugs are greater than their individual activities. Indifference is when the combined action is equal to the sum of their independent activity. Antagonism is when the sum is less than the parts.

Category: Pharmacology Notes